Biocatalysis with Thermostable Enzymes: Structure and Properties of a Thermophilic ‘ene’‐Reductase related to Old Yellow Enzyme

Abstract

We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

Identification and characterization of a novel “thermophilic-like” Old Yellow Enzyme from the genome of Chryseobacterium sp. CA49

“…2). In addition, residues involved in subunit interactions in members of "thermophilic-like" subfamily [12,20] are well conserved in Chr-OYE3 (Fig. 2).…”

“…modulation of catalytic potential, FMN binding, substrate binding and subunit interactions in OYE members were also conserved in Chr-OYE3 and exhibited similar amino acid preference with members in the "thermophilic-like" subfamily. The modulator of redox potential of "classical" OYEs is considered to be a strictly conserved threonine, such as Thr37 in OYE1 [19], while the equivalent residue in "thermophilic-like" members is a cysteine [12,13,18,20], which is also conserved in Chr-OYE3 (Fig. 2).…”

“…Subsequently, three thermostable OYEs from thermophilic organisms have been proved to be members in this subfamily [3,13,20,21]. They are CrS from Thermus scotoductus SA-01, GkOYE from Geobacillus kaustophilus and TOYE from Thermoanaerobacter pseudethanolicus E39.…”

Structural insights into stereospecific reduction of α, β-unsaturated carbonyl substrates by old yellow enzyme from Gluconobacter oxydans

“…The different oligomerization forms are determined by the sequence variable regions, which are involved in the formation of the potential oligomerization interface. 2,38) Gox0502 forms monomers in solution as determined by analytic ultracentrifugation (AUC) assay (Fig. S1).…”

Two “classical” Old Yellow Enzymes from Chryseobacterium sp. CA49: Broad substrate specificity of Chr-OYE1 and limited activity of Chr-OYE2

“…Another highly conserved residue Y376 in OYE1 that is known to form hydrogen bonding with substrates and inhibitors is well conserved in Chr-OYE1 and Chr-OYE2 as well. However, the highly conserved histidine pair involved in substrate positioning (via hydrogen binding) and the catalytic tyrosine (proposed proton donor) [31,32] were only found in Chr-OYE1, but not in Chr-OYE2. The second histidine and the catalytic tyrosine have been respectively replaced by alanine and methionine in Chr-OYE2.…”